1999 Fiscal Year Annual Research Report

細菌における蛋白質および染色体DNAの動的局在性の機構

Research Project

Project/Area Number	11694221
Research Category	Grant-in-Aid for Scientific Research (A)
Research Institution	Kumamoto University
Principal Investigator	平賀壯太熊本大学, 医学部, 教授 (40027321)
Co-Investigator(Kenkyū-buntansha)	山添光芳熊本大学, 医学部, 助手 (00284745) 仁木宏典熊本大学, 医学部, 講師 (70208122)
Keywords	DNAメチレーション / 複製起点 / 複製開始 / DnaA / SeqA / 染色体分配
Research Abstract	我々はパリ大学ジャンクモノ研のM.Kohiyama博士、パリ大学B.Holland博士と共同研究を行った。His-tagを結合したSeqAを大量にニッケルカラムで精製し、そのSeqAを使って、oriCプラスミドのin vitro複製系におけるSeqAの阻害効果、およびoriCとの結合様式を詳細に解析した。その結果次のことが明らかになった。(i)大腸菌のSeqA蛋白質は複製直後の半メチル化GATC配列に特異的に結合し、染色体DNAの複製調節に関与する。(ii)SeqAは半メチル化oriCDNAのin vitro複製系を強く阻害する。(iii)半メチル化DNAの場合はoriC領域の4つのDnaA boxおよびAT-rich、13-mer領域に結合するが、全メチル化DNAの場合は、4つのDnaA boxesのみに結合する。(iv)DnaAは半メチル化DNAの場合にはDnaA boxesの他にAT-rich、13-mer領域にも結合し、全メチル化DNAの場合には4つのDnaA boxesのみに結合する。(v)半メチル化DNAにDnaAを結合させた後に、SeqAを加えると約3/4のDnaAはDNAから遊離する。即ち、複製後の半メチル化oriCに結合していたDnaAがSeqAによって遊離することが複製開始阻害の原因の1つとなっていると結論できる(投稿中)。Kohiyama博士は1ヶ月間来日して平賀研で共同研究を行い細胞内における複製直後のDNAを観察する顕微鏡法を確立した。E.Boye博士を熊本大に招待しセミナーをやってもらい、さらにKohiyama博士らと共に三木市で開催された3R Simpojiumにおいて共同研究の成果を発表した。J.-M.Louarn博士が分離した逆変異を使いFISH法で染色体の動態を明らかにした(Niki et al.,2000)。

Research Products
(14 results)

All Other

All Publications (14 results)

[Publications] Niki H.: "Dynamic organization of chromosomal DNA in Escherichia coli."Genes Dev. 14. 212-213 (2000)
[Publications] Niki H.: "Progressing molecular cell biology in bacteria: chromosome partitioning and cell division"蛋白質・核酸・酵素. 45. 115-117 (2000)
[Publications] Iwasaki,H.: "Identification of functional motifs of the RuvB motor protein for Holliday Junction branch migration an AAA+ class helicase, by mutational analyses and sequence alignment of the homologs."Mol. Microbiol.. (in press). (2000)
[Publications] Teff,D.: "A colicin-tolerant Escherichia coli mutant that confers Hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH(HflB)."FEMS Microbiol. Lett.. 183. 115-117 (2000)
[Publications] Makino,S.: "Second transmembrane segment of FtsH plays a role in its proteolytic activity and homo-oligomerization."FEBS Lett.. 460. 554-558 (1999)
[Publications] Karata,K: "Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases: site-directed mutagenesis of the ATP-dependent protease FtsH."J. Biol. Chem.. 274. 26225-26232 (1999)
[Publications] Landoulsi A.: "Initiation of DNA replication in Δcya mutants of Escherichia coli K12."Biochimie. 81. 827-834 (1999)
[Publications] d'Alencon E.: "Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli."Biochimie. 81. 841-846 (1999)
[Publications] Lyngstadaas A.: "The gene for 2-phosphogiycolate phosphatase (gph) in Escherichia coli is located in the same operon as dam and at least five other diverse genes."Biochim Biophys Acta. 1472. 367-384 (1999)
[Publications] Reshetnyak E.: "Hemi-methylated oriC DNA binding activity found in non-specific acid phosphatase"Mol Microbiol. 31. 167-175 (1999)
[Publications] Onogi T.: "The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli."Mol Microbiol. 31. 1775-1782 (1999)
[Publications] Niki H.: "Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system."Mol Microbiol. 34. 498-503 (1999)
[Publications] Yamazoe M.: "Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli."EMBO J. 18. 5873-5884 (1999)
[Publications] Carlson C. R.: "Regulation of the start of DNA replication in Schizosaccharomyces pombe"J Cell Sci. 112. 939-946 (1999)

1999 Fiscal Year Annual Research Report

細菌における蛋白質および染色体DNAの動的局在性の機構

Principal Investigator

平賀 壯太 熊本大学, 医学部, 教授 (40027321)

Research Products

[Publications] Niki H.: "Dynamic organization of chromosomal DNA in Escherichia coli."Genes Dev. 14. 212-213 (2000)

[Publications] Niki H.: "Progressing molecular cell biology in bacteria: chromosome partitioning and cell division"蛋白質・核酸・酵素. 45. 115-117 (2000)

[Publications] Iwasaki,H.: "Identification of functional motifs of the RuvB motor protein for Holliday Junction branch migration an AAA+ class helicase, by mutational analyses and sequence alignment of the homologs."Mol. Microbiol.. (in press). (2000)

[Publications] Teff,D.: "A colicin-tolerant Escherichia coli mutant that confers Hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH(HflB)."FEMS Microbiol. Lett.. 183. 115-117 (2000)

[Publications] Makino,S.: "Second transmembrane segment of FtsH plays a role in its proteolytic activity and homo-oligomerization."FEBS Lett.. 460. 554-558 (1999)

[Publications] Karata,K: "Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases: site-directed mutagenesis of the ATP-dependent protease FtsH."J. Biol. Chem.. 274. 26225-26232 (1999)

[Publications] Landoulsi A.: "Initiation of DNA replication in Δcya mutants of Escherichia coli K12."Biochimie. 81. 827-834 (1999)

[Publications] d'Alencon E.: "Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli."Biochimie. 81. 841-846 (1999)

[Publications] Lyngstadaas A.: "The gene for 2-phosphogiycolate phosphatase (gph) in Escherichia coli is located in the same operon as dam and at least five other diverse genes."Biochim Biophys Acta. 1472. 367-384 (1999)

[Publications] Reshetnyak E.: "Hemi-methylated oriC DNA binding activity found in non-specific acid phosphatase"Mol Microbiol. 31. 167-175 (1999)

[Publications] Onogi T.: "The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli."Mol Microbiol. 31. 1775-1782 (1999)

[Publications] Niki H.: "Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system."Mol Microbiol. 34. 498-503 (1999)

[Publications] Yamazoe M.: "Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli."EMBO J. 18. 5873-5884 (1999)

[Publications] Carlson C. R.: "Regulation of the start of DNA replication in Schizosaccharomyces pombe"J Cell Sci. 112. 939-946 (1999)

平賀壯太熊本大学, 医学部, 教授 (40027321)